1. Field of the Invention
The present invention relates to a novel method of detecting specific nucleic acid sequences as well as a detecting reagent and kit therefor.
2. Related Art
The isolation of enzymes that can join separate DNA molecules was fundamental to the development of recombinant DNA technology. Using these enzymes, called ligases, genetic elements derived from different genomes can be brought to be expressed together in an organism. Ligase-assisted reactions have also come to play an increasing role in gene detection. The principle employed in such reactions is that two probe-segments, complementary to target sequences situated in juxtaposition, are joined to a contiguous probe sequence through the agency of a DNA ligase. This is in contrast to more conventional detection schemes where the hybridization of single probes to target sequences is detected after removal of free probe molecules.
One type of ligase-assisted detection method is disclosed in U.S. Pat. No. 4,988,617, which relates to an assay for determining the nucleic acid sequence in a region of a nucleic acid test substance which has a known possible mutation in at least one target nucleotide position of the sequence. The assay comprises annealing two oligonucleotide probes to immediately adjacent segments of a substantially complementary test DNA or RNA molecule which contains the possible mutation(s) near the segment joint, and adding a linking agent, usually a ligase. The conditions are selected such that when the target nucleotide is correctly base paired, the probes are covalently joined, and if not correctly base paired due to a mismatching nucleotide(s) near the the segment joint, the probes are incapable of being covalently joined by the ligase. The presence or absence of linking is detected as an indication of the sequence of the target nucleotide.
A similar ligase-assisted detection method is disclosed in EP-A-185 494. In this method, however, the formation of a ligation product depends on the capability of two adjacent probes to hybridize under high stringency conditions rather than on the requirement of correct base-pairing in the joint region for the ligase to function properly as in the above U.S. Pat. No. 4,988,617. Other references relating to ligase-assisted detection are, e.g., EP-A-330 308, EP-A-324 616, EP-A-473 155, EP-A-336 731, U.S. Pat. No. 4,883,750 and U.S. Pat. No. 5,242,794.
The principal advantages of ligase-based detection strategies over other molecular genetic detection reactions are at least three-fold:
(i) The reaction is inherently very specific. Ligation reactions are based on the coincidence of two separate probe sequences on a target sequence, and this is unlikely to occur in the absence of the appropriate target molecule even under non-stringent reaction conditions. For this reason the reaction is suitable in standardized, automated schemes. PA1 (ii) The ligation reaction can be employed to assess the accuracy of hybridization at positions immediately surrounding the junction between two probes. Due to the substrate requirements of ligases, terminally mismatched probes are ligated at a substantially reduced rate. In this manner, allelic sequence variants can be distinguished. PA1 (iii) The act of ligation creates a unique molecule, not previously present in the-assay. This circumstance can be employed, for example, by taking advantage of the increased hybridization stability upon ligation. PA1 a) providing a detectable probe, which optionally may be branched and which has two nucleic acid end parts at least partially complementary to and capable of hybridizing to two at least substantially neighbouring regions of the target sequence, PA1 b) hybridizing the probe to the target sequence under hybridizing conditions, PA1 c) covalently connecting the ends of the hybridized probe with each other to form a circularized-structure, which interlocks with the target strand through catenation, PA1 d) subjecting the target sequence to non-hybridizing conditions and/or exonuclease activity to remove any non-circularized probe from the target sequence, thereby retaining only the circularized probe bound to the target molecule, PA1 e) optionally repeating steps b) to d) one or more times, and PA1 f) detecting the presence and, if desired, location of remaining probe as indicative of the presence of the target nucleic acid sequence.
JP-A-4262799 and JP-A-4304900 both disclose the use of ligation reactions combined with amplification reactions for detecting a target nucleic acid sequence in a specimen sample. The methodology comprises contacting the sample in the presence of a ligase with a straight chain probe polynucleotide, which has a sequence designed to be cyclized, or circularized, as the result of the presence of a target nucleic acid sequence. The cyclized polynucleotide is then used as a template in an enzymatic polymerization reaction. By adding a primer which is at least partially complementary to the cyclized probe together with a nucleic acid polymerase and nucleotide triphosphates, a single stranded chain nucleic acid is formed which has a repeated sequence complementary to the probe and at least partially to the template. The amplification product is then detected either via a labelled nucleic acid triphosphate incorporated in the amplification, or by an added labelled nucleic acid probe capable of hybridizing to the amplification product.
While this prior art technique comprises a ligase-based detection step, which as discussed above, per se may provide for high specificity, the specificity of the method will rather depend on the specificity in the generation and detection of the amplified product.